3. Research Associate- 1999 - 2001: Council of Scientific and Industrial Research New Delhi, India.

Contribution to the scientific advancement

1. We have designed series of dominant negative (DN) inhibitor proteins against B-ZIP transcription factors involved in seed formation and maturation. These DN inhibits the DNA binding activity of B-ZIP53, B-ZIP10, B-ZIP25 known to be master regulators of seed maturation. Our designed protein inhibitors are excellent tools for studying signal transduction pathways where biological redundancy is common.

2. Transgenic Arabidopsis expressing one of the designed dominant negative A-ZIP53 gave aborted seed phenotype. Using Immuno-precipitation and advance Mass spectrometry we identified all interacting B-ZIP transcription factors involved in seed formation. Numbers of them are novel and their involvement in seed formation and maturation is reported for the first time. A logical extension of this work is to identify seed interactome of fruit crops like guava, kinnow and melon where seedlessness is highly desirable. Once identified gene editing tools like CRISPR/Cas may be used to edit seed-specific B-ZIP transcription factors leading to small/seedless phenotype.

3. At National Institutes of Health (NIH), USA, biotechnology tools developed by us are widely used by researchers all over the world. For example A-SREBP, a dominant negative of SREBP family of transcription factor has proven to be an excellent tool for studying lipid and cholesterol metabolism in animals. This along with other plasmids is available from Addgene at nominal charges.

4. At NABI, one of our endeavours is to develop protein expression vectors for bacterial as well as mammalian host systems. In this direction we have developed a highly efficient pET system that utilizes a designed N-terminal tag to enhance the expression of any gene in E. Coli. Furthermore, we have developed a Ligation Independent Cloning (LIC) plasmid that enables us to clone genes in high-throughput manner. Also using LIC reduces the cloning time to just 5 minutes and saves on costly restriction enzymes and ligases.

5. In epigenetics field we are the first on to prove that DNA methylation of cytosine on CG dinucleotide of a transcription factor binding site is not always repressive. At least in few tissue-specific genes in mouse model and differentiating primary tissue culture methylation of cytosine is required for differentiation of fibroblasts to adipocytes. We showed that methylation is a switch for differentiation. Unmethylated CREB binding site (TGACGTCA) is bound by CREB B-ZIP protein and is required to keep cells in undifferentiated state. Methylated cytosine cannot be bound by CREB but now can be bound by C/EBP family of B-ZIPs, later binding is required for fibroblasts to differentiate into adipocytes.

Current area of research:

1. Basic biology of seed development

In plants seed development is a complex and poorly understood phenomenon. In fruit crops like kinnow and guava small seed/seedlessness is highly desirable therefore studying the biology of seed development is both challenging and rewarding. Among others, numbers of seed-specific genes are regulated by Basic leucine zipper (B-ZIP) family of transcription factors (TFs). Our research group is focused on deciphering the roles that these dimeric B-ZIP TFs play in seed-specific gene regulation. Situation get complicated due to biological redundancy, a common phenomenon among plant’s B-ZIP TFs, where a gene knockout does not give a corresponding phenotype and success of conventional methodology of gene manipulation like CRISPR/Cas or siRNA is limited. One such example is b-ZIP53 that along with its heterodimerizing partners, namely b-ZIP10 and b-ZIP25 play a pivotal role in regulating genes involved in seed development and maturation. Interestingly insertion lines of all three b-ZIPs set viable seeds. To address this problem we have adapted a novel protein-based gene knockdown technology. In an unprecedented studies in plants we designed a dominant-negative protein called A-ZIP53 (here A stand for acidic extension) that in addition to targeting b-ZIP53 also inhibits b-ZIP10 and b-ZIP25.

An ultimate aim of our research is to design proteins that are biologically active. In this direction we made a transgenic Arabidopsis that constitutively expresses A-ZIP53. Number of T2 lines showed prominent seed phenotype. Seed were unviable and showed an aborted embryo phenotype. This proves the efficacy and supremacy of our technology in studying a biologically phenomenon that is dictated by number of closely related transcription factors.

To have an insight into mechanistic basis of aborted seed phenotype we used mass spectrometry to identify novel interacting partners of A-ZIP53. Using silique samples, A-ZIP53 with T7 tag was immunoprecipitated (IP) and its interacting partners were unravelled by NanoLC-MS-MS. It is for the first time that we identified hitherto unknown partners of B-ZIP53 namely, b-ZIP69, 29, 14, 34, and 46 that may play role in seed development. In future homologs of these seed-specific b-ZIP TFs will be studied in fruit crops like kinnow and guava. We hope that our study will help in the development of seedless fruits.

Transcription factor (TF) binding to cis-element of a promoter is often the first step in cascade of events that ultimately results in gene expression. In addition to “consensus sequence” a TF can bind to number of ‘sub-optimal’ sites in genome many of them are biologically relevant. Presently, only ~1% of binding preferences are known for thousands of identified transcription factors making DNA binding landscape of TFs an underexplored area of research. The problem is more pronounced in plants, more so in bread wheat where knowledge of binding sites of seed-specific B-ZIP TFs is completely lacking.

In addition to classical methods like footprinting and EMSA there is a spurt of high-throughput techniques like SELEX (systematic evolution of ligands by exponential enrichment), bacterial one-hybrid system (B1H) to study genome-wide DNA-binding sites for a specific TF with range of affinities. In our lab we have adapted Bind-N-Seq methodology to study in vitro DNA-binding of a single B-ZIP TF to thousands of binding sites available in a genome. Technique involves the binding of TF pure protein to double-stranded 90 mer oligo. Since all reactants are in solution phase there are no issues of steric hindrance and system closely follows perfect thermodynamic conditions. We have cloned 10 seed-specific B-ZIPs from bread wheat and expressed them to 95+ homogeneity. 40 oligos libraries are constructed that will be used for binding experiments. Next generation sequencing will be used to create binding landscape for each B-ZIP TF under defined experimental conditions.

3. Direct Protein transfection studies as a tool to understand differentiation of Pre-adipocyte to adipocyte: A spatial and temporal study of gene regulation during adipogenesis.

CCAAT/enhancer-binding proteins (C/EBP) are a family of b-ZIP transcription factors that have diverse roles in regulation of pre-adipocytes differentiation. We are interested in regulating the binding of C/EBP to its targets sites in promoters of adipogenesis genes responsible for pre-adipocyte differentiation. Induction of pre-adipocytes with differentiation inducers leads to the rapid and transient increase in the expression of C/EBPβ and C/EBPδ. It leads to the Mitotic Clonal Expansion (MCE) of cells, a phenomenon unique to pre-adipocyte. MCE ceases after two days of induction and is coincident with transcriptional activation of C/EBPα and Peroxisome proliferator-activated receptor γ (PPARγ). We are using a designed dominant negative protein (A-C/EBP) that can interact with all family members of C/EBPs. We are in an envious position where adipogenesis can be studied in real time by transfecting 3T3-L1 pre-adipocytes cell line with A-C/EBP. Presently we are designing and cloning different versions of A-C/EBP protein.